Production and use of diels alder adducts of vinyl porphyrins, of metal complexes thereof, and of compositions containing such adducts and complexes

ABSTRACT

Families of Diels Alder adducts and of metal complexes of Diels Alder adducts, which are useful as particularly active compounds for use in photodynamic therapy, are disclosed. The Diels Alder adducts and a preferred family of metal complexes have the structures of Formulas 1, 2, 3 and 4, below: ##STR1## where R1, R2, R3 and R4 can be the same or different, and each is methyl, ethyl or an amino acid moiety which is a part of an amide produced by reaction between an amine function of a naturally occurring amino acid and a carbonyl function of the adduct, R5, R6 and R7 can be the same or different, and each is ethyl or an amino acid moiety which is a part of an amide produced by reaction between an amine function of a naturally occurring amino acid and a carbonyl function of the adduct.

REFERENCE TO RELATED APPLICATIONS

This is a continuation in part of application Ser. No. 08/321,387, filedOct. 11, 1994 is pending as a continuation in part of application Ser.No. 07/912,079, filed Jul. 8, 1992 now U.S. Pat. No. 5,354,858, as acontinuation of application Ser. No. 07/677,408, filed Mar. 28, 1991 nowabandoned.

FIELD OF THE INVENTION

This invention relates to the production and use of new Diels Alderadducts of vinyl porphyrins, to the production and use of metalcomplexes of these adducts, to the production and use of compositionscontaining such adducts and metal complexes, and to a method fordetecting and destroys diseased tissue which involves administering aDiels Alder adduct or metal complex to a human or animal patient. Afterthese compounds are administered, they localize preferentially indiseased tissue; after they have been administered, and have localizedin diseased tissue, their presence can be detectead by illumination withultra violet or light of a wavelength at which they have an absorbancepeak, causing them to fluoresce. They can also be used to treat diseasedtissue; after they have been administered and have localized in diseasedtissue, illumination with visible light having a wavelength at whichthey show an absorbance peak causes a reaction which involves theformation of singlet oxygen, and which damages or destroys the diseasedtissue. Specifically, the new Diels Alder adducts, which are useful asparticularly active compounds for use in photodynamic therapy, have thestructures of Formula 1 and Formula 2, below: ##STR2## where R1, R2, R3and R4 can be the same or different, and each is methyl or ethyl, and R8is an alkyl group other than t-butyl having from one to four carbonatoms. The new metal complexes of the foregoing Diels Alders adductshave the structure of Formula 3, Formula 4, Formula 5, Formula 6,Formula 7 or Formula 8, below: ##STR3## where R1, R2, R3, R4, R5, R6 andR7 can be the same or different, and each is an alkyl group other thant-butyl having from one to four carbon atoms,

an alkylene group having from 2 to 4 carbon atoms,

a group having the formula R₂ N(R₃)₂ where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; R₃ is hydrogen or an alkyl group having from 1 to2 carbon atoms and the two R₃ groups can be the same or different,

an amino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct,

a monoclonal antibody moiety which is attached to the adduct moietythrough a carbonyl which is a part of an amide produced by reactionbetween an amine function of a monoclonal antibody and a CO₂ R', CH₂ CO₂R' or CH₂ CH₂ CO₂ R' group of the adduct, and wherein the moiety is of amonoclonal antibody which selectively binds to malignant tumors,

a group having the formula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; A is a physiologically acceptable anion; and R₄ isan alkyl group having from 1 to 2 carbon atoms and the three R₄ groupscan be the same or different,

a group having the formula R₂ OH where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond,

an ester having the structure CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R',where R' is hydrogen or an alkyl group other than t-butyl having from 1to 4 carbon atoms,

R8 is an alkyl group other than t-butyl having from one to four carbonatoms, and

M comprises a metal cation that is complexed with two of the nitrogensof the adduct and is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In,La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb, Tc-99m, Th,Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr.

Preferred families of Diels Alder adducts and metal complexes accordingto the invention have a substituent which is an amino acid moiety whichis a part of an amide produced by reaction between an amine function ofa naturally occurring amino acid and a carbonyl function of the adduct.These families have the structures of Formulas 9, 10, 11 and 12, below:##STR4## In formulas 9, 10, 11 and 12, R1, R2, R3 and R4 can be the sameor different, and each is methyl, ethyl or an amino acid moiety which isa part of an amide produced by reaction between an amine function of anaturally occurring amino acid and a carbonyl function of the adduct,R5, R6 and R7 can be the same or different, and each is ethyl or anamino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct, and R8 is an alkyl group other thant-butyl having from one to four carbon atoms, with the proviso that oneof R1, R2, R3, R4, R5, R6 and R7 is an amino acid moiety.

Formula 1, where R8 is methyl, is reproduced below, with the numbers 1through 12 added to identify some of the carbon atoms in the Diels-Alderadduct of Formula 1; the same numbering is used herein to identify thecorresponding carbon atoms in the Diels-Alder adduct of Formula 2 and inthe metal complexes of Formulas 3 through 8 (this is not theconventional numbering used in porphyrin chemistry, where numbers areassigned to all the carbons in the nucleus). The carbons that arenumbered in the following formula are those which are capable of beingsubstituted in the parent porphyrin. The R1, R2, R3 and R4 substituentsare on the 1, 4, 7 and 10 carbon atoms while the ethyl substituents areon the 2, 8, and 11 carbon atoms, and the six-membered exocyclic ring isfused to the 4 and 5 carbon atoms. ##STR5##

DISCUSSION OF RELATED ART

Various modified porphyrins which appear green because they absorb lightin the orange-red range of wavelengths are disclosed in "Levy et al."(U.S. Pat. No. 4,883,790, granted Nov. 28, 1989 for WAVELENGTH-SPECIFICCYTOTOXIC AGENTS; a "modified porphyrin" is sometimes called a Gp in thepatent). Levy et al. also discloses conjugates of the modifiedporphyrins and of hematoporphyrin ("Hp") with receptor ligands which arecapable of binding to cell surfaces and with immunoglobulins orimmunologically reactive portions of immunoglobulins. The conjugates canbe composed, the patent states, of modified porphyrins or Hp covalentlybonded to receptor ligands, immunoglobulins or immunologically reactiveimmunoglobulin portions or of modified porphyrins covalently bonded tolinking moieties which are in turn covalently bonded to the receptorligands, immunoglobulins or immunologically reactive immunoglobulinportions. The preferred modified porphyrins (and the only ones that arespecifically disclosed) are "obtained using Diels-Alder reactions withporphyrin nuclei under conditions which effect a reaction at only one ofthe two available conjugated, nonaromatic diene structures present inthe protoporphyrin-IX nucleus". (column 3, lines 4 and following). Levyet al. also states (column 3, lines 36 et seq.):

"Specific preparation of compounds useful in the invention is describedby Morgan, A. R., et al., J Chem Soc Chem Commun (1984) 51:1094. Asdescribed in these publications, protoporphyrin-IX dimethyl ester, whenreacted with strong Diels-Alder dienophile reagents such astetracyanoethylene, is derivatized to the dihydro-dibenzo derivatives.However, when more weakly electron withdrawing groups are utilized onthe Diels-Alder reagent, hydromonobenzo derivatives are formed. Thus,there are obtained compounds shown as formulas 1 and 2 of FIG. 1 whereinR¹ and R² represent the original Diels-Alder reagent substituents and R³represents the substituents natively or originally on the porphyrinnucleus."

Protoporphyrin IX dimethyl ester has the following structure: ##STR6##The patent specifically discloses six modified porphyrins (formulas 1-6of FIG. 1) all of which retain one of the vinyl groups of theprotoporphyrin IX dimethyl ester, so that the ethyl substituent on the 2carbon in the compounds of the instant invention is vinyl in three ofthe modified porphyrins of the patent. In the other three modifiedporphyrins, the exocyclic ring is fused to the 1 and 2 carbons and thereis a vinyl substituent on the 5 carbon. In all six of the modifiedporphyrins, there is a methyl substituent on the 11 carbon, where thecompounds of the instant invention have an ethyl substituent.

The Levy et al. patent also reports the assessment of the "efficacy ofthe conjugates and of the Gp compounds of the invention in vivo" (column11, lines 5 and 6) by tests that are identified, and includes Table 4,which gives test data or Hp, for two Hp conjugates (one with "C-Mab"which is called an "irrelevant monoclonal preparation" and one with"B16G antibody"), for a mixture of B16G antibody and Hp and or twocontrols: phosphate buffered saline and B16G antibody, stating thatsimilar results "are obtained for Gp alone or Gp conjugates". Table 4gives, among other data, the percent of animals that were tumor freeafter 100 days; this percentage ranges from 12.5 to 43 for five of theHp conjugates tested, and is zero for the Hp conjugate with C-Mab, orall of the compositions which contained Hp or Hp plus B16G antibody, andor the controls. The Levy patent neither discloses nor suggests metalcomplexes of the Diels Alder adducts with which it is concerned.

Certain porphyrins and families of purpurins and chlorins and metalcomplexes thereof and the use of the purpurins, chlorins, metalcomplexes and porphyrins in the manner described above for the detectionand treatment of tumors are all known. For example, PCT/US86/02824discloses certain purpurins, chlorins, and metal complexes thereof, andtheir use for the detection and treatment of tumors. In addition,European patent application EP142,732 is said (C.A. 103: 123271S) todisclose certain chlorins of a different family and that they accumulatepreferentially in the cancer cells of hamsters infected with pancreaticcancer.

Further, a chemical mixture derived from hematoporphyrin, calledhematoporphyrin derivative, and often abbreviated "HpD", can beadministered intravenously and used in the manner described above forthe detection and treatment of tumors. Hematoporphyrin can be producedfrom protoporphyrin IX, a porphyrin that can be separated from blood.HpD is a mixture of many different porphyrins and related compounds, theexact composition not being fully own (see, for example, PorphyrinPhotosensitization, edited by David Kassel and Thomas J. Dougherty,Plenum Press, New York and London, 1983, pp.3-13). As a consequence, thechlorins and purpurins of PCT/US86/02824 are preferred over HpD for thisuse because they are single, own compounds. In addition, the chlorinsand purpurins have absorbance peaks at longer wavelengths and showgreater absorbances, by comparison with HpD; the longer wavelength peaksare advantageous because light of the longer wavelengths is capable ofgreater penetration of tissue, while the greater absorbances aredesirable because less light energy is required to cause a given degreeof reaction.

The production of the nickel complex of an octa-ethyl benzochlorin hasbeen disclosed (Arnold et al., J. C. S. PERKIN I, pages 1660-1670,1979). The complex is produced by reaction in dry NN-dimethylformamidebetween phosphorus oxychloride and nickel meso-vinyl octaethylporphyrin.The major product reported was nickel 5-(β-Formylvinyl)octaethylporphyrin (80 percent yield); in addition, the authors reporteda 5 percent yield of the nickel benzochlorin and a 15 percent yield of ademetallated product (which was not a benzochlorin). The nickeloctaethylbenzochlorin has been found to be substantially inert insofaras the ability to cause a cytotoxic response is concerned.

The production of a verdin isomer mixture by refluxing a mesorhodinisomer mixture in acetic acid has been reported (The Porphyrins, VolumeII, pages 137 and 138, edited by David Dolphin, Academic Press, NewYork, San Francisco and London, 1978). Woodward et al. J.A.C.S., 1960,82, p. 3800 and Morgan, J. Org. Chem., 1986, 51, 1347 disclose that theporphyrin derivatives form when purpurins stand in sunlight in thepresence of air. U.S. Pat. No. 4,878,891 ("Judy et al.", granted Nov. 7,1989) discloses the sterilization of blood and other body fluids andtissues, using either HpD or a composition containing about 90 percentof dihematoporphyrin ether as a photosensitizer. The photosensitizer isadministered, e.g., intravenously, 5 to a blood donor or a patient, and,after a suitable time, a blood or the like sample is removed from thedonor or patient, and is irradiated with light of a suitable wavelength.Alternatively, the photosensitizer is added to a sample of blood or thelike, and the sample is irradiated after a suitable time.

U.S. Pat. Nos. 5,093,349 ("Pandey et al.", Mar. 3, 1992) and 5,079,262("Kennedy et al.", Jan. 7, 1992) disclose both the systemic and thetopical administration of photosensitizers and irradiation with light ofa suitable wavelength.

HpD, under the trivial designation "porfimer sodium" has undergoneclinical testing in humans, and has been approved in Canada for use inphotodynamic therapy of superficial bladder carcinoma, in TheNetherlands for use in such treatment of certain lung and esophagealcancers, and in Japan for use in such treatment of early stage lungcancer, superficial esophageal cancer, superficial and early stagegastric cancers, and early stage cervical cancer, including cervicaldysplasia. In the United States, the Oncology Drugs Advisory Committeeof the Food and Drug Administration has recommend approval of porfimersodium as a palliative treatment for totally obstuctive and somepartially obstructive cancers of the esophagus (Seminars in Oncology,Vol. 21, No. 6, Suppl 15 (December), 1994: pp 1-3, W. B. SaundersCompany).

Earlier publications (see, for example, published InternationalApplication WO 84/01382, Apr. 12, 1984 and Porphyrin Photosensitization,edited by David Kassel and Thomas J. Dougherty, Plenum Press, New Yorkand London, 1983, pp.3-13 and cited references) disclose the use of HpDto treat tumors in various animals, including DBA₂ Ha/D mice and ICRSwiss (Albino) mice in which tumors had been transplanted, and alsoincluding pet cats and dogs.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention is a family of Diels-Alder adducts which have thestructure of one of Formulas 1 and 2, above, where R1, R2, R3 and R4 canbe the same or different, and each is methyl or ethyl. The invention isalso a family of metal complexes of Diels Alder adducts having thestructure of one of Formulas 3, 4, 5, 6, 7 and 8, above, and a methodfor detecting and treating diseased tissue in a human or animal patientwhich depends upon the characteristic of the Diels Alder adducts andmetal complexes that they bind to diseased tissue, but are rejected bynormal tissue. The method involves administering intravenously,intramuscularly, subcutaneously, intraperitoneally or topically aneffective amount of one of the Diels-Alder adducts or metal complexes tothe patient, and, after sufficient time for healthy tissue Diels Alderadduct or metal complex, irradiation of the relevant region of thepatient. For detection, the irradiation can be with ultra violet orvisible light of a wavelength at which the Diels Alder adduct or metalcomplex has an absorbance peak; during the irradiation, the patient isexamined for fluorescence, which will occur if there is residual DielsAlder adduct or metal complex. For treatment, the irradiation is withvisible light of a wavelength at which the Diels Alder adduct or metalcomplex has an absorbance peak, and is of such intensity and durationthat it causes at least one reaction and the destruction of the diseasedtissue. Examples of the types of diseased tissue that can be treatedinclude malignant tumors and lesions, e.g., of the vagina and bladder,and such cutaneous lesions as are involved 5 in proriasis.

The present inventors coauthored with others a paper which was publishedon Apr. 1, 1990, (Journal of Medicinal Chemistry, 33, pages 1258 et seq.[1990]) disclosing, inter alia, the preparation of two Diels-Alderadducts according to the instant invention and their efficacy in thetreatment of transplantable FANFT-induced rat bladder tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples constitute the best modes presently contemplatedby the inventors, but are presented solely to illustrate and disclosethe invention, and are not intended to be limiting.

As used herein, and in the appended claims, the terms "percent" and"parts" refer to percent and parts by weight, unless otherwiseindicated; g means gram or grams; mg means milligram or milligrams; ngmeans nanogram or nanograms; pg means picogram or picograms; cm meanscentimeter or centimeters; mm means millimeter or millimeters; L meansliter or liters; mL means milliliter or milliliters; μL means microliteror microliters; m/o means mole percent, and equals 100 times the numberof moles of the constituent designated in a composition divided by thetotal number of moles in the composition; v/v means percent by volume;w/v means weight per unit of volume, and is in terms of g/L; M meansmolar and equals the number of gram moles of a solute in one liter of asolution; μM means micromolar and equals the number of microgram molesin one liter of a solution; mM means millimolar and equals the number ofmilligram moles of a solute in one liter of a solution; N means normal,and equals the number of gram equivalents of a solute in one liter ofsolution; and μN means micronormal and equals the number of microgramequivalents of a solute in one liter of solution. All temperatures arein °C., unless otherwise indicated.

Example 1 describes the production of a Diels-Alder adduct ("Adduct I")of 2-vinyl-3,7,8,12,13,17,18-heptaethylporphyrin ("Porphyrin I"; Chang,C. K. et al., J. Org. Chem. 52, 926 [1987]) from Porphyrin I anddimethyl acetylenedicarboxylate. Adduct I has the structure of Formula 1where R1, R2, R3 and R4 are ethyl. Porphyrin I has the followingstructure, which is a general formula for vinyl porphyrins which can beused to produce Diels Alder adducts according to the invention. InPorphyrin I, R1, R2, R3 and R4 are ethyl: ##STR7##

EXAMPLE 1

Adduct I was synthesized from a solution of 20 mg Porphyrin I and 1 mLdimethyl acetylenedicarboxylate in 30 mL toluene. The solution washeated under reflux for about 120 hours until an absorbance band of thePorphyrin I at 624 nm disappeared and an absorbance band appeared at 653nm. The solution was cooled; the solvent was removed under reducedpressure; and the residue was chromatographed on silica gel usingdichloromethane containing 2v/v diethyl ether and 2v/v toluene as theeluent. A red band (first) and a green band (second) were collected. Thesolvent was removed from the red band under reduced pressure; and theresidue was recrystallized from a dichloromethane-methanol solvent,yielding 7 mg Porphyrin I. The solvent was removed from the green bandunder reduced pressure; and the residue was recrystallized from adichloromethane-methanol solvent, yielding Adduct I (30 percent oftheory), which was identified by ¹ H NMR spectroscopy; λ_(max) 651, 594,534, 499, 400 (ε25201, 3046, 5999, 7062, 75951).

Example 2 describes the preparation of2-vinyl-7,12,17-triethyl-3,8,13,18-tetramethyl-porphyrin ("Porphyrin II"and the production of a Diels-Alder adduct ("Adduct II") of Porphyrin IIand dimethyl acetylenedicarboxylate. Adduct II has the structure ofFormula 1 where R1, R2, R3 and R4 are methyl. Porphyrin II has theforegoing general formula for vinyl porphyrins from which Diels Alderadducts according to the invention can be produced. In Porphyrin II, R1,R2, R3 and R4 are methyl. The preparation of Porphyrin II from2,3-Dihydroxy -2,7,12,17-tetra-ethyl-3,8,13,18 -tetramethylchlorin("Chlorin I"; Chang et al., J. Org. Chem. 1987, 52, 926) is describedfirst. Chlorin I has the following structure, which is general fordihydroxy chlorins from which vinyl porphyrins can be produced; inChlorin I, R1, R2, R3 and R4 are methyl: ##STR8##

EXAMPLE 2 Preparation of Porphyrin II

Porphyrin II was prepared from 25 mg Chlorin I by reaction withphosphorus pentoxide for five hours at 140°. The phosphorus pentoxideand a 25 mL beaker which contained the Chlorin I were placed in a vacuumoven which was maintained at a pressure of 10 mm during the reaction.After the reaction, the solid in the beaker was removed from the oven,and cooled. The soluble portion was then dissolved in the minimum amountof dichloromethane. The mixture which resulted was chromatographed onsilica gel, using 60v/v hexane in dichloromethane as the eluent.

Production of Adduct II

Adduct II was synthesized front a solution of 20 mg Porphyrin II and 1mL dimethyl acetylenedicarboxylate in 30 mL toluene. The solution washeated under reflux for about 96 hours until the absorption spectrumindicated that the Porphyrin II had all reacted. Two bands, one of whichwas identified by NMR spectroscopy as Adduct II, were recovered frontthe crude product by chromatography.

Example 3 describes the chemical shift of Adduct I to produce AdductIII, a compound having the structure of Formula II where R1, R2, R3 andR4 are ethyl, and the chemical shift of Adduct II to produce Adduct IV,a compound having the structure of Formula II where R1, R2, R3 and R4are methyl.

EXAMPLE 3

Solutions containing, in one case, 10 mg Adduct I and a few drops oftriethanolamine in 10 mL dichloromethane is refluxed for two hours and,in a second case, 10 mg Adduct II and a few drops of triethanolamine in10 mL dichloromethane are refluxed for two hours. The solvent and excesstriethanolamine are then removed in vacuo, and the crude product ispurified by chromatography, producing almost quantitative yields ofAdduct III and of Adduct IV.

Adduct I and Adduct II were used in in vivo testing conducted on maleFischer CDF(F344)/CrlBr rats weighing 135 to 150 g in whose flanks twotransplantable FANFT-induced rat bladder tumors (AY27) had been graftedsubcutaneously. (Use of this system is reported by Sehnan, S. H., etal., Cancer Research, pp. 1924-1927, May, 1984.) When the tumors reachedone cm in transverse diameter the animals were injected with sensitizer.

The two adducts were dissolved in a commercially available non-ionicsolubilizer and emulsifier obtained by reacting ethylene oxide withcastor oil in a ratio of 35 moles of ethylene oxide per mole of castoroil, diluting the resulting solution with 1,2-propanediol, and producingan emulsion with the resulting solution and 0.9 percent aqueous sodiumchloride solution. The specific non-ionic solubilizer used is availablefrom BASF under the designation CREMOPHOR EL; it is composed of fattyacid esters of polyglycols, glycerol polyglycols, polyethylene glycolsand ethoxylated glycerol. The test solutions were prepared from 50 mgportions of each of the adducts, about 1 mL warm solubilizer (enough todissolve the test compound), and enough 1,2-propanediol to make asolution of the adduct in a mixed diol/solubilizer solvent containing32.9 percent solubilizer; finally, enough 0.9 percent aqueous sodiumchloride was added to make 10 mL test solution so that the finalconcentration of the adduct in the test solution was 5 mg per mL. Eachtest solution was made, with mechanical shaking and stirring, bydissolving the adduct in the solubilizer, diluting the resultingsolution with the indicated amount of 1,2-propanediol, and adding thesodium chloride solution to the diluted solution. A control solution wasalso prepared for use with each test solution. The control was identicalwith the test solution except that it contained no adduct.

The testing involved injecting each rat with a solution of the adduct,dosage 5.0 mg per kg of body weight in one series of tests and 1.0 mgper kg of body weight in another, or with the same volume of theappropriate control, irradiating one of the two tumors while the otherwas shielded from light, sacrificing the animals, and examining thetumors. The injections were made via the dorsal tail vein. Theirradiation of one of the tumors occurred twenty four hours after eachrat was injected. The tumors were examined twelve days after treatment.

Tumor temperature and body core temperature were monitored, usingthermistors, one placed into the tumor and one placed intrarectally.Tumor temperature was kept within 2° of body core temperature bydirecting a jet of cool air over the tumor.

The light source used for irradiation was a slide projector that had a500 watt bulb filled with a red filter which is available from CorningGlass Works under the designation 2418. The light was reflected 90° by asilvered mirror, and was focused onto the tumor with a secondarycondensing lens. The light intensity on the tumor was monitored, using aphotometer/radiometer that is available from United Detector Technologyunder the designation "UDT #351", and was maintained at 200 mw per cm².

Six rats were injected with each of the adduct test solutions and twowere injected with the appropriate control solution.

Twelve days after the irradiation, none of the treated tumors of therats that had been injected with 5.0 mg per kg of body weight of eitheradduct could be detected either by palpation or histologically, but theuntreated tumors and those in the rats that had been injected with thecontrol had continued to grow. The rats to which 1.0 mg per kg of bodyweight of the adducts had been administered were sacrificed by anintracardiac injection of saturated aqueous potassium chloride solution,and the control and treated tumors were harvested and desiccated toconstant weight. One hundred times the dry weight of the tumors of thetreated rats divided by the dry weight of the tumors of the control ratswas zero for the rats treated with Adduct I and 7 for those treated withAdduct II. During the testing, the rats were under barbituate anesthesia(65 mg per kg body weight).

None of the irradiated tumors of the rats that were treated with 1.0 mgper kg of body weight of Adduct I and only fifty percent of theirradiated tumors of the rats that were treated with that dose of AdductII could be detected palpably.

The production of Adduct I and of Adduct II by reaction between dimethylacetylene-dicarboxylate and Porphyrin I and Porphyrin II is described inexamples 1 and 2, respectively. The reaction of these examples isgeneral in the sense that it can be used to prepare Diels Alder adductsof other vinyl porphyrins which have the structure shown above. Suchvinyl porphyrins are either known, or can be produced by the methoddescribed in Example 2 for the preparation of Porphyrin II fromdihydroxy chlorins having the foregoing structure. The requireddihydroxy chlorins are either known or can be produced by OsO₄ oxidationof the corresponding porphyrins, which are either known or can beproduced by known reactions from the requisite dipyrrolic intermediates,e.g., dipyrromethanes and dipyrromethenes, which, in turn are eitherknown or can be synthesized from the requisite pyrroles. The requisitepyrroles, if not available, can be synthesized by the classical KnorrReaction and variations, and by other known reactions, and can bemanipulated and transformed (see, for example, David Dolphin, ThePorphyrins, Volume I, Structure and Synthesis, Part A, Academic Press,New Your, San Francisco and London, 1978, pages 101-163. The pyrroleshave the following structure: ##STR9## where A can be H, CH₃, an ester,a nitrile, a cyanovinyl or an amide group, G can be H, an ester, anitrile, a cyanovinyl or an amide group and B and C are substituentswhich appear in the ultimate porphyrin, frequently lower alkyl groups,particularly methyl and ethyl.

Dipyrrolic intermediates, e.g., dipyrromethanes and dipyrromethenes, canbe synthesized from pyrroles, and can be converted to porphyrins byknown reactions; some porphyrins can be synthesized directly frompyrroles (see, for example, David Dolphin, supra, pages 85-100 and163-234). Dipyrromethanes and dipyrromethenes have the followingstructures. ##STR10##

By way of example, "Octamethylporphyrin" can be synthesized by heating3,4-dimethylpyrrole (foregoing structure, where A is HOOC, B and C areCH₃ and D is CH₂ OH) at 160-170° and "Octaethylporphyrin" can besynthesized by heating 3,4-diethylpyrrole, where A is HOOC, B and C areCH₂ CH₃ and D is CH₂ OH. Porphyrins can also be produced fromdipyrromethanes by way of an aldehyde coupling reaction, a formic acidor orthoformate ester condensation, by the "dialdehyde synthesis" or bythe Vilsmeier pyrroketone synthesis, and from dipyrromethenes by theFischer synthesis, or by reaction with dipyrromethanes. The porphyrinsthat are produced have the following structure where R is hydrogen andR1 through R4 and R5 through R8 have the same meanings as B, C, E and Fin the dipyrromethane and dipyrromethene starting materials when theporphyrins are synthesized front these precursors: ##STR11## Inoctamethylporphyrin and octaethylporphyrin, R is hydrogen and R1 throughR8 are methyl in the former and ethyl in the latter.

Example A describes the preparation of2,3-dihydroxy-2,3,7,8,12,13,17,18-octaethylchlorin ("Chlorin II"; Changet al, supra) from a solution of 1.168 g octaethylporphyrin and 1 mLpyridine in 250 mL dichloromethane and 1.0 g osmium tetroxidc in 10 mLdiethyl ether. Chlorin II has the foregoing general formula fordihydroxy chlorins where R1, R2, R3 and R4 are ethyl.

EXAMPLE A

The octaethylporphyrin/pyridine solution is mixed with the osmiumtetroxide and ether, and the reaction mixture which results is stirredat room temperature of about 22° for two days. The reaction mixture isthen diluted with 50 mL of methanol, and H₂ S is bubbled through thediluted mixture for 15 minutes. Osmium sulfide, which is precipitated bythe H₂ S, is then separated by filtration, and the solvent is evaporatedfrom the filtrate. The residue is triturated with methanol, whichdissolves the Chlorin II, leaving the octaethylporphyrin. The Chlorin IIis further purified on a silica gel column using dichloromethanecontaining 0.5v/v methanol. The method of the first paragraph of Example2 can then be used to synthesize Porphyrin I from Chlorin II.

It is known that metal complexes of purpurins and chlorins, particularlythe tin and zinc complexes, are more effective compounds for use inphotodynamic therapy than the corresponding metal-free compounds. It iscontemplated that the metal complexes of the adducts according to theinstant invention will also be more effective, and that they can beproduced by the procedures used to prepare the purpurins and chlorins.Example B, below, illustrates the method contemplated for thepreparation of such complexes.

EXAMPLE B Production of Sn Diels Alder Adduct I

A solution is prepared by dissolving 20 mg Diels Alder Adduct I in 20 mLacetic acid and 100 mg tin chloride is added to the solution; themixture which results is refluxed for about 24 hours until theelectronic spectrum of the reaction mixture indicates that chelation iscomplete. The reaction mixture is then concentrated to 7 mL and allowedto cool to room temperature of about 22° . Product which precipitates isrecovered by filtration, dissolved in a mixed solvent composed of 5 mLdichloromethane and 2 mL hexane, and recrystallized, yielding the Sncomplex of Porphyrin I, which has the structure of Formula 3, supra,where R1 through R7 are ethyl, R8 is methyl, and M is Sn.

The procedure of Example B can be used to produce metal complexes ofother adducts according to the invention. Specifically, an equivalentamount of Adduct II can be substituted for the Adduct I, or zincacetate, cobalt acetate, silver acetate, palladium acetate, or platinumacetate can be substituted for the tin chloride, or both substitutionscan be made. In this manner, metal complexes of Diels Alder adductshaving the structure of Formula 3 or 4 where M is Sn, Co, Ag, Pd, Pt orZn can be produced from Diels Alder adducts having the structure ofFormula 1 or 2.

Other complexes can be produced by the method of Example B from saltscontaining cations other than acetate, and producing complexes whichhave the structures of Formulas 3 and 4, but where M does not representmerely a metal cation. Examples of salts that can be substituted forzinc acetate in the Example B procedure are given below, together withthe identity of M in the foregoing FIGS.:

    ______________________________________                                        Salt                Identity of M                                             ______________________________________                                        FeCl.sub.3          Fe(Cl)                                                    MnCl.sub.4          Mn(Cl)                                                    InCl.sub.3          In(Cl)                                                    VCl.sub.4 *         V(O)                                                      Tl(CF.sub.3 CO.sub.2).sub.3                                                                       Tl(OAc)(H.sub.2 O)                                        SnCl.sub.2          Sn(OH).sub.2                                              [Rh(CO).sub.2 Cl].sub.2                                                                           Rh(Cl)(H.sub.2 O)                                         ______________________________________                                         *Using phenol as the solvent instead of glacial acetic acid.             

The procedure of Example B can also be modified by substituting phenolfor glacial acetic acid and metal chelates of pentane, 2,4-dione forzinc acetate to produce complexes of any of the Diels Alders adducts.Metals that can be so reacted (as pentane, 2,4-dione chelates) and theidentity of M in the complex that is produced are set forth in thefollowing table:

    ______________________________________                                        Metal    Identity of M                                                                              Metal     Identity of M                                 ______________________________________                                        Al       Al(acac)*    Th        Th(acac).sub.2                                Sc       Sc(acac)     U         U(acac).sub.2                                 Ga       Ga(acac)     La        La(acac).sub.2                                In       In(acac)     Ce        Ce(acac)                                      Mo       Mo(acac)     Nd        Nd(acac)                                      Ti       Ti(acac).sub.2                                                                             Sm        Sm(acac)                                      Zr       Zr(acac).sub.2                                                                             Gd        Gd(acac)                                      Hf       Hf(acac).sub.2                                                                             Tb        Tb(acac)                                      Eu       Eu(acac)     Dy        Dy(acac)                                      Pr       Pr(acac)     Ho        Ho(acac)                                      Yb       Yb(acac)     Er        Er(acac)                                      Y        Y(acac)      Tm        Tm(acac)                                      Lu       Lu(acac)                                                             ______________________________________                                         *The pentane, 2,4dione portion of a chelate thereof with a metal.        

Complexes of the Diels Alder adducts can also be produced by theprocedure of Example B, substituting dimethylformamide for glacialacetic acid and CrC₁₂ for zinc acetate. Metal complex formation occursat higher temperatures when dimethylformamide is used, because of itshigher boiling temperature. M in the complexes is Cr(OH).

Similarly, complexes of the Diels Alder adducts can be produced by theprocedure of Example B, substituting pyridine for glacial acetic acidand PbCl₂ for zinc acetate. M in the complexes is Pb.

Example C, below, describes the production of a Diels Alder adduct from500 mg Protoporphyrin IX Dimethyl ester dissolved in 50 mL dry tolueneand 0.5 mL diethyl acetylenedicarboxylate (see Pangka et al., J. Org.Chem, 1986, 51, 1094-1100).

EXAMPLE C

A reaction mixture composed of the diethyl acetylenedicarboxylate andthe Protoporphyrin IX Dimethyl ester solution is refluxed in the dark atroom temperature of about 22° for six days. The solvent is then removedin vacuo and the residue is chromatographed on SiO₂ with dichloromethanecontaining 2v/v diethyl ether. Two isomers, "Adduct V" and "Adduct VI",are recovered. The two adducts have the structures shown below, where Ris ethyl: ##STR12## The procedure of Example 3 can be used o cause achemical shift of Adduct V to Adduct VII and of Adduct VI to AdductVIII, compounds having the structures shown below, where R is ethyl:##STR13## Adduct VII and Adduct VIII can be selectively reduced bytreatment with hydrogen in the presence of palladium on charcoal (seeLevy et al., supra), to produce Adduct IX and Adduct X, which have thefollowing structures, where R is ethyl: ##STR14## Metal complexes ofAdducts V and VI can be produced by the procedures of Example B and themodifications thereof discussed above, producing Complex V and ComplexVI, which have the following structures, where R is ethyl and M is Ag,Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb,Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn, Tb, Tc-99m, Th, Ti, Tl, Tin, U, V, Y,Yb, Zn or Zr: ##STR15## Similarly, metal complexes of Adducts VII andVIII can be produced by the procedures of Example B and themodifications thereof discussed above, producing Complex VII and ComplexVIII, which have the following structures where R is ethyl and M is Ag,Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb,Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn, Tb, Tc-99m, Th, Ti, TI, Tin, U, V, Y,Yb, Zn or Zr: ##STR16##

Finally, metal complexes of Adducts IX and X can be produced by theprocedures of Example B and the modifications thereof discussed above,producing Complex IX and Complex X, which have the following structureswhere R is ethyl and M is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf,Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn, Tb,Tc-99m, Th, Ti, TI, Tin, U, V, Y, Yb, Zn or Zr: ##STR17##

Where any of R1 through R8 of any of the foregoing adducts or adductmetal complexes has a free CO₂ H group, that moiety can be reacted withan amino acid moiety, which can be a monoclonal antibody, to form anamide. Example D is illustrative of such reactions:

EXAMPLE D

A Diels Alder adduct coupled to a monoclonal antibody is produced from(1) 20 mg Diels Alder Adduct metal complex produced as described abovewhere one of R1 through R7 is CO₂ H, CH₂ CO₂ H or CH₂ CH₂ CO₂ Hdissolved in 1.25 ml water and 0.8 ml N,N-dimethyl formamide, (2) 20 mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. HC1 dissolved in 0.6 mlwater and (3) 15 mg monoclonal antibody dissolved in 5 ml distilledwater. The Adduct solution is added to the carbodiimide hydrochloridesolution, and the combined solution is mixed with the monoclonalantibody solution. After 30 minutes, the reaction is quenched by adding0.05 ml monoethanol amine, and the conjugated material, i.e., the amideof the monoclonal antibody and the Adduct, is dialyzed exhaustively at4° against 0.001N phosphate buffered saline, pH 7.4.

The procedure of Example D is generally applicable to couple proteinsand amino acids which, as in the example, can be monoclonal antibodiesto Diels Alder Adducts and metal complexes thereof having the structuresof formulas 1 through 8 where one of R1 through R8 is a CO₂ H or thelike group. The amino acid so coupled, using the Example D procedure,when not a monoclonal antibody, is preferably a naturally occurringamino acid, most desirably lysine, histidine, arginine, cystine, serine,aspartic acid, aspartic acid esters, glutamic acid and glutamic acidesters. Five of these amino acids have the formula ##STR18## where R hasthe meaning indicated below: ##STR19## The formula for aspartic acid isgiven below: ##STR20## The preferred aspartic acid and glutamic acidesters are esters of lower alkyl alcohols, most desirably those otherthan t-butyl having from 1 to 4 carbon atoms.

In the procedures described above, Diels Alder adducts were produced byreactions between a vinyl compound and either dimethylacetylencdicarboxylate or diethyl acetylenedicarboxylate. Otheracetylenedicarboxylates, e.g., ones where the two alkoxy groups can bethe same or different, and each has the formula R8O- where R8 is analkyl group other than t-butyl having from one to four carbon atoms, canbe substituted, so that the two R8 groups in the foregoing formulas canbe the same or different, and each can be an alkyl group other thant-butyl having from one to four carbon atoms.

The Diels Alder adducts and complexes can be administered topically, forexample as dilute, e.g., 1 percent w/w solutions in DMSO or ethanol tonon-malignant lesions, e.g., of the vagina or bladder, or to suchcutaneous lesions as are involved in psoriasis, followed by illuminationof the area involved with light of a wavelength at which the Diels Alderadduct or complex has an absorbance peak. The adduct or the likesolution should be applied only to the lesions to prevent damage tohealthy tissue adjacent the lesions. Illumination of the lesions, forexample, for from 15 to 30 minutes then completes the treatment. It isto be understood, however, that Diels Alder adducts and complexesaccording to the invention can also be administered systemically, i.e.,intravenously, intramuscularly or subcutaneously, in the treatment ofnon-malignant lesions.

The production of Diels Alder adduct and complex solutions in thespecific non-ionic solubilizer that is available under the designationCREMOPHOR EL, and the production of emulsions of such solutions with1,2-propanediol and saline solution is described above, as is the use ofsuch solutions to detect and treat tumors. It will be appreciated thatadducts and metal complexes can be dissolved in other non-ionicsolubilizers and that the solutions can be used to produce emulsionsthat can be administrated intravenously, intramuscularly orsubcutaneously. For example, other reaction products of ethylene oxideand castor oil can be so used, as can reaction products of ethylene,propylene and other similar oxides with other fatty acids and thereaction products of propylene and other similar oxides with castor oil.Similarly, glycols other than 1,2-propanediol can be used in producingthe emulsions for intravenous and other administration, or the glycolcan be omitted, particularly if the solubilizer is prepared to have alower viscosity and greater compatibility with water, by comparison withthe solubilizer that is available under the designation CREMOPHOR EL. Itis necessary only that the solution or emulsion be one which isphysiologically acceptable and of a suitable concentration, or dilutableto a suitable concentration, for intravenous or other administration orfor local administration, should that be desirable. An indefinitelylarge number of such solutions and emulsions will be apparent to thoseskilled in the relevant art from the foregoing specific disclosure.Similarly, the aqueous phase need not be 0.9 percent w/w or any otherconcentration of sodium chloride. Such saline is presently favored forintravenous administration, but other aqueous phases can also be used,so long as the entire composition is physiologically acceptable forintravenous or other administration and, in fact, other aqueous phasesmay subsequently be favored. Indeed, other aqueous phases or organicphases may also be favored for local administration.

Dosages of 1 mg and of 5 mg per kg of body weight were used in the invivo procedures described above. It has been determined only that thebiological consequences described above were caused by the dosagesadministered, not that any dosage reported is either a minimum or amaximum. It will be appreciated, therefore, that it is necessary only touse an effective amount of a Diels Alder adduct or metal complexaccording to the invention in the detection and treatment of tumors andother diseased tissue, preferably as small a dosage as possible, andthat the exact dosage can be determined by routine experimentation. Bothsystemic administration, specifically intravenous, intramuscular andsubcutaneous, and local administration, i.e., as a lotion or a solutionin dimethyl sulfoxide or ethanol, have been described above; however, itwill also be appreciated that other methods of administration will besuitable, at least in some instances. In addition, the Diels Alderadducts and metal complexes can be used in vitro to eradicate infectiouspathogenic biological contaminants from blood, semen and other bodyfluids or from tissue, e.g., skin, removed from a human or animalpatient or donor. Pathogenic contaminants that can be eradicated includeenvelope-containing viruses, bacteria, malarial, trypanosomes and otherparasites. A Diels Alder adduct or metal complex according to theinvention, e.g., at a dosage of 1 or 5 mg per kg of body weight, asdescribed above, can be administered to a patient or a donor and, aftera suitable time, a blood, semen, skin or the like sample can be takenand irradiated with light of a suitable wavelength, either as taken inthe case of a fluid, or suspended in a physiologically acceptable salinesolution in the case of a tissue. Instead, a blood, semen, skin or thelike sample can be taken and a Diels Alder adduct or a metal complexaccording to the invention added thereto, either as taken in the case ofa fluid, or suspended in a physiologically acceptable saline solution inthe case of a tissue and, after a suitable time for the Diels Alderadduct or metal complex to associate itself with the pathogeniccontaminants, the sample can be irradiated with light of a suitablewavelength. When a Diels Alder adduct or metal complex according to theinvention is added to a fluid sample, whether a body fluid or asuspension of a tissue in a saline solution, the dosage, in either case,should be sufficient for there to be enough of the Diels Alder adduct ormetal complex to associate with all of the pathogenic contaminants inthe sample, and usually ranges from about 0.1 to 50 mg per L of sample,preferably from about 2 1to 50 mg per L. The wavelength of theirradiating light should be or should include one at which the DielsAlder adduct or metal complex has an absorbance peak. The density ofradiation used with the sample can range from 0.1 to 50 Joules per cm²,preferably from 1 to 20 Joules per cm², and most desirably about 5Joules per cm².

The use of HPD and a compound which is said to contain about 90 percentof DHE (dihematoporphyrin ether) to treat blood, semen and the like bodyfluids and skin and other body tissues is disclosed in detail in "Judyet al." (U.S. Pat. No. 4,878,891, issued Nov. 7, 1989). Diels Alderadducts and metal complexes according to the present invention are shownby the experimental results reported above to "bind" in the same way asthe two materials disclosed by Judy et al., and, when they areilluminated by light of a suitable wavelength, to undergo a reactionwhich destroys the cell or diseased tissue to which they are bound,differing in that they are more effective as photosensitizers.Accordingly, the disclosure of Judy et al. is applicable to thetreatment of blood and other body fluids and skin and other body tissueswith Diels Alder adducts and metal complexes according to the instantinvention, except that the dosages and light intensity required aresomewhat less with the adducts and metal complexes of the instantinvention. Indeed, the present Diels Alder adducts and metal complexescan be substituted, generally, for other sensitizers for photodynamictherapy, taking into account the comparative effectiveness of the DielsAlder adducts and metal complexes and of the sensitizer for which theyare substituted. The comparative effectiveness can be determined byinjecting various dosages of the two sensitizers in test animals, andascertaining what dosage of each is required to achieve a givenbiological result.

Illumination of tumors containing a Diels Alder adduct or a metalcomplex in accordance with the instant invention can be a surfaceillumination with a conventional light source, as described above, orcan be a surface illumination with a laser. The illumination can also beinto the body of a tumor, for example through optical fibers insertedthereinto.

It will be appreciated that various changes and modifications arepossible from the specific details of the invention as described abovewithout departing from the spirit and scope thereof as defined in thefollowing claims.

We claim:
 1. A method for detecting diseased tissue in a living mammalwhich comprises administering intravenously, intramuscularly,subcutaneously or topically to a human or animal patient an effectiveamount of a Diels Alder adduct or of a metal complex of a Diels Alderadduct and, after healthy tissue has rejected the Diels Alder adduct orthe metal complex, examining the patient under illumination which causesresidual Diels Alder adduct or metal complex to fluoresce, wherein theDiels Alder adduct has the structure of Formula 9 or of Formula 10, andthe metal complex of a Diels Alder adduct has the structure of Formula 3or of Formula 4: ##STR21## where M comprises a metal cation that iscomplexed with two of the nitrogens of the adduct and is Ag, Al, Ce, Co,Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt,Rh, Sb, Sc, Sm, Sn, Tb, Tc-99m, Th, Ti, T1, Tm, U, V, Y, Yb, Zn orZr,R1, R2, R3 and R4 can be the same or different, and each is methyl,ethyl, an amino acid moiety which is a part of an amide produced byreaction between an amine function of a naturally occurring amino acidand a carbonyl function of the adduct, or a monoclonal antibody moietywhich is attatched to the adduct moiety through a carbonyl which is apart of an amide produced by reaction between an amine function of amonoclonal antibody and a CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' group ofthe adduct, and wherein the moiety is of a monoclonal antibody whichselectively binds to malignant tumors, R5, R6 and R7 can be the same ordifferent, and each is ethyl, an amino acid moiety which is a part of anamide produced by reaction between an amine function of a naturallyoccurring amino acid and a carbonyl function of the adduct, or amonoclonal antibody moiety which is attatched to the adduct moietythrough a carbonyl which is a part of an amide produced by reactionbetween an amine function of a monoclonal antibody and a CO₂ R', CH₂ CO₂R' or CH₂ CH₂ CO₂ R' group of the adduct, and wherein the moiety is of amonoclonal antibody which selectively binds to malignant tumors, and R8is an alkyl group other than t-butyl having from one to four carbonatoms.
 2. A method as claimed in claim 1 for detecting a tumor wherein ametal complex of a Diels Alder adduct having the structure of Formula 3or Formula 4 is administered.
 3. A method as claimed in claim 2 fordetecting a tumor wherein M of the metal complex of the Diels Alderadduct is Sn or Zn.
 4. A method for treating a living human or animalpatient who has diseased tissue, which method comprises administering tothe patient, intravenously, intramuscularly, subcutaneously ortopically, an effective amount of a Diels Alder adduct or of a metalcomplex of a Diels Alder adduct and, after healthy tissue has rejectedthe Diels Alder adduct or the metal complex, irradiating the diseasedtissue with light of a wave length which causes a reaction whichdestroys the diseased tissue, wherein the Diels Alder adduct has thestructure of Formula 9 or of Formula 10, and the metal complex of aDiels Alder adduct has the structure of Formula 3 or of Formula 4:##STR22## where M comprises a metal cation that is complexed with two ofthe nitrogens of the adduct and is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga,Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn,Tb, Tc-99m, Th, Ti, TI, Tm, U, V, Y, Yb, Zn or Zr,R1, R2, R3 and R4 canbe the same or different, and each is methyl, ethyl, an amino acidmoiety which is a part of an amide produced by reaction between an aminefunction of a naturally occurring amino acid and a carbonyl function ofthe adduct, or a monoclonal antibody moiety which is attatched to theadduct moiety through a carbonyl which is a part of an amide produced byreaction between an amine function of a monoclonal antibody and a CO₂R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' group of the adduct, and wherein themoiety is of a monoclonal antibody which selectively binds to malignanttumors, R5, R6 and R7 can be the same or different, and each is ethyl,an amino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct, or a monoclonal antibody moiety whichis attatched to the adduct moiety through a carbonyl which is a part ofan amide produced by reaction between an amine function of a monoclonalantibody and a CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' group of the adduct,and wherein the moiety is of a monoclonal antibody which selectivelybinds to malignant tumors, and R8 is an alkyl group other than t-butylhaving from one to four carbon atoms.
 5. A method as claimed in claim 4for treating a patient wherein a metal complex of a Diels Alder adducthaving the structure of Formula 3 or Formula 4 is administered.
 6. Amethod as claimed in claim 5 for treating a patient wherein M of themetal complex of a Diels Alder adduct is Sn or Zn.